For many years it was generally believed that the production of reactive oxygen species (ROS) was an unwanted byproduct of aerobic metabolism and other cellular enzymatic processes and that ROS were uniformly deleterious in nature. We have spent the majority of our energies pursuing an alternative hypothesis; that production of ROS are tightly regulated, the targets of ROS are specific, and that oxidants contribute to disease progression, at least in part, through the redox-regulation of specific pathways (See Finkel and Holbrook, Nature, 408, 2000, 239-247; Finkel, T., Current Opinions Cell Biol., 15; 2003, 247-254; Balaban, Nemoto and Finkel, Cell 120, 2005, 483-497). [unreadable] [unreadable] Nearly ten years ago we observed that certain cells produce high levels of ROS when stimulated by peptide growth factors (Sundaresan et al, Science 1995, 270 296-299). The production of ROS was transient, peaking in the first few minutes following ligand stimulation and returning to baseline within 30 minutes after stimulation. Our initial observation was in vascular smooth muscle cells stimulated with the growth factor PDGF, but subsequently it has become clear that similar events transpire in a wide variety of different cell types stimulated by a host of different ligands. Interestingly we found that inhibiting this rise in ROS blocked the initial signaling events demonstrating an essential role in ROS generation for normal physiological signal transduction. [unreadable] We have also previously demonstrated that the source of the ligand stimulated ROS-generator in non-phagocytic cells shared certain molecular and biochemical similarities with the phagocytic NADPH oxidase. In particular, we were able to demonstrate a role for the small GTPase Rac1 in the regulation of the intracellular redox state. We have also shown with the help of our collaborators that the related GTPase Ras also plays an important role in redox regulation within cells. Interestingly, the ability of Ras proteins to induce transformation in the context of immortalized cell, or to induce senescence in the context of primary cells, appears dependent in some fashion on the ability of Ras proteins to induce a change in the level of ROS.[unreadable] [unreadable] These observations have been extended recently in our lab in an attempt to understand the molecular regulators of mitochondrial ROS production as well as mitochondrial oxygen consumption. My laboratory has a significant focus on the molecular basis of mammalian aging and age-related diseases and in particular the role that mitochondrial metabolism and oxidative stress plays in these processes. In this regard we have focused on three areas during this last year. The first area of emphasis is to explore the biology of mammalian sirtuins. This is a family of NAD-dependent deacetylases first implicated in aging of lower organisms. The founding member of this family is the yeast Sir2 gene, first described as a regulator of transcriptional silencing and later as a modulator of life span. In mammals, seven sirtuin family members (Sirt1-7) exist. We have predominantly concentrated on the biology of Sirt1, the closest mammalian homologue of the yeast Sir2 gene. We have shown that in mice, Sirt1 expression is induced upon starvation via a pathway involving the Foxo3a transcription factor and p53 (Nemoto et al., 2004). We have further demonstrated a role for Sirt1 in regulating mitochondrial flux. This process we believe involves the generation of new mitochondria through PGC-1-dependent biogenesis (Nemoto et al., 2005) as well as the clearance of old mitochondria through autophagy (Lee et al., 2008). We have also explored the biology of mitochondrial Sirt3, demonstrating a role for this protein in overall ATP homeostasis and in particular, in regulating the acetylation of the electron transport chain (Ahn et al., PNAS, in press).[unreadable] [unreadable] A second aim of the laboratory is to understand the role of stem cells in aging. Our interest in this area was sparked by our previous clinical observations regarding a decline in endothelial cell progenitor number and function in patients with impaired vascular function (Hill et al., 2003). We were interested in understanding the potential biological connections between aging and stem cell dysfunction. Our major new finding with regard to this aim involved a mouse model of accelerated aging known as the klotho mouse. This animal model lacks the gene encoding klotho that encodes for a circulating protein. We demonstrated that klotho mice have widespread stem and progenitor cell dysfunction and that klotho can serve as a circulating Wnt inhibitor (Liu et al., 2007). This result suggests a novel potential role for Wnt signaling in mammalian aging. While we continue to pursue these observations, we have also explored other models of stem cell dysfunction including the bmi1 deficient mouse. This animal is characterized by a post-natal defect in hematopoietic and neural stem cell selfrenewal. We have recently demonstrated that tissues within the bmi1-/- mice are characterized by a rise in intracellular reactive oxygen species (ROS) and that this oxidative stress can activate the DNA damage response (DDR) pathway. Genetic ablation of the DDR by Chk2 deletion can extend the life span of the bmi1-/- mice (Liu et al, submitted). [unreadable] [unreadable] Finally we have pursued the connection between mTOR activity and mitochondrial metabolism. The mTOR pathway serves as a nodal point for energy homeostasis and has been implicated in mediating the beneficial effects of caloric restriction and overall lifespan for a variety of simple organisms. We have demonstrated that this pathway is also implicated in mammalian mitochondrial metabolism (Schieke et al., 2006). These observations complement our observations with another mammalian longevity gene product p66shc (Nemoto et al., 2006; Smith et al., 2005). We have also recently demonstrated novel connections between mitochondrial function, mTOR activity, cell cycle progression and stem cell fate (Schieke et al., Cell Cycle, in press; Schieke et al., JBC, in press).